A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence

Huntingtin encodes a 3144 amino acid protein, with a polyglutamine repeat tract at the N-terminus. Expansion of this repeat tract above a pathogenic threshold of 36 repeats is the causative mutation of Huntington's disease, a neurodegenerative disorder characterized by loss of striatal neurons. Here we have characterized twenty Huntingtin commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.


Introduction
Huntington's Disease (HD) is a neurodegenerative disorder inherited in an autosomal dominant manner, presenting with a spectrum of progressive motor, cognitive, and psychological impairments, typically with adult-onset of symptoms. 1lthough the HD causative gene, HTT, was discovered over three decades ago, there are still no disease-modifying treatments available for patients, and progress unpicking the molecular pathology of the disease remains slow. 2 HD arises from a heterozygous expansion mutation of the trinucleotide CAG repeat tract in exon 1 of HTT, located on chromosome 4, above a critical threshold of ~36 repeats.This mutation results in expansion of the polyglutamine stretch at the N-terminus of the 3144 amino acid Huntingtin protein.Huntingtin functions as a scaffold protein, engaging in extensive protein-protein interactions, 3 forming various multi-protein complexes to carry-out its diverse array of functions.Modulation of this interaction network by the polyglutamine expansion contributes to degeneration within the central nervous system, affecting medium spiny neurons at the onset of disease. 4,5e low expression level, complex interactome and large size of the 348 kDa Huntingtin protein have given rise to technical challenges which have hindered precise determination of its molecular function, or how this is altered in disease.0][11] Here we evaluated the performance of twenty commercial antibodies for Huntingtin for use in western blot, immunoprecipitation, and immunofluorescence, enabling biochemical and cellular assessment of Huntingtin properties and function.The platform for antibody characterization used to carry out this study was endorsed by a committee of industry and academic representatives.It consists of identifying human cell lines with adequate target protein expression and the development/contribution of equivalent knockout (KO) cell lines, followed by antibody characterization procedures using most commercially available antibodies against the corresponding target protein.The standardized consensus antibody characterization protocols are openly available on Protocol Exchange (DOI: 10.21203/rs.3.pex-2607/v1). 12e authors do not engage in result analysis or offer explicit antibody recommendations.A limitation of this study is the use of universal protocols -any conclusions remain relevant within the confines of the experimental setup and cell line used in this study.Our primary aim is to deliver top-tier data to the scientific community, grounded in Open Science principles.This empowers experts to interpret the characterization data independently, enabling them to make informed choices regarding the most suitable antibodies for their specific experimental needs.Guidelines on how to interpret antibody characterization data found in this study are featured on the YCharOS gateway. 13

Results and discussion
Our standard protocol involves comparing readouts from WT (wild type) and KO cells. 14,15The first step is to identify a cell line(s) that expresses sufficient levels of a given protein to generate a measurable signal.To this end, we examined the DepMap transcriptomics database to identify all cell lines that express the target at levels greater than 2.5 log 2 (transcripts per million "TPM" + 1), which we have found to be a suitable cut-off (Cancer Dependency Map Portal, RRID: SCR_017655).We generated an HTT KO line in DMS 53 as it expresses the endogenous HTT transcript at 6.1 log 2 (TPM+1), which is above the average range of cancer cell lines analyzed.A commercial HAP1 HTT KO is also available; HAP1 expresses HTT at 3.7 log 2 (TPM+1) RNA level.A HEK293T HTT KO cell line has been developed and used elsewhere 16 (Table 1).All three cell line backgrounds were evaluated by western blot using a high-performing Huntingtin antibody detected in Figure 1.DMS 53 was identified as the most suitable cell line (Figure 2), which can be explained by its high expression of the HTT transcript compared to other two cell lines.Thus, DMS 53 WT and KO cell lines were generated and used to evaluate the antibodies in all applications.
For western blot experiments, WT and HTT KO protein lysates were ran on SDS-PAGE, transferred onto nitrocellulose membranes, and then probed with twenty Huntingtin antibodies in parallel (Table 2, Figure 1).We then assessed the capability of all twenty antibodies to capture Huntingtin from DMS 53 protein extracts using immunoprecipitation techniques, followed by western blot analysis.For the immunoblot step, a specific Huntingtin antibody identified previously (refer to Figure 1) was selected.Equal amounts of the starting material (SM), the unbound fraction (UB), as well as the whole immunoprecipitate (IP) eluates were separated by SDS-PAGE (Figure 3).
For immunofluorescence, twenty antibodies were screened using a mosaic strategy.First, DMS 53 WT and HTT KO cells were labelled with different fluorescent dyes in order to distinguish the two cell lines, and the Huntingtin antibodies were evaluated.Both WT and KO lines were imaged in the same field of view to reduce staining, imaging and image analysis bias (Figure 4).Quantification of immunofluorescence intensity in hundreds of WT and KO cells was performed for each antibody tested, 12 and the images presented in Figure 3 are representative of this analysis.
In conclusion, we have screened twenty commercial Huntingtin antibodies by western blot, immunoprecipitation, and immunofluorescence by comparing the signal produced by the antibodies in human DMS 53 WT and HTT KO cells.
Several high-quality and renewable Huntingtin antibodies were identified in all applications.Researchers who wish to study Huntingtin in a different species are encouraged to select high-quality antibodies, based on the results of this study, and investigate the predicted species reactivity of the manufacturer before extending their research.
The underlying data for this study can be found on Zenodo, an open-access repository for which YCharOS has its own collection of antibody characterization reports. 17 Thank you to the Structural Genomics Consortium, a registered charity (no.1097737), for your support on this project.The Structural Genomics Consortium receives funding from Bayer AG, Boehringer Ingelheim, Bristol-Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute (grant no.OGI-196), the EU and EFPIA through the Innovative Medicines Initiative 2 Joint Undertaking (EUbOPEN grant no.875510), Janssen, Merck KGaA (also known as EMD in Canada and the United States), Pfizer and Takeda.
An earlier version of this of this article can be found on Zenodo (DOI: 10.5281/zenodo.11582780).
The article pointed out the current technical difficulties in studying Huntington's Disease and identified the differences in antibodies to the protein Huntingtin, used throughout the HD research community, as a particular problem.The study formed part of the wider YCharOS collaboration to characterise commercial antibodies for human proteins using standardized protocols with the intention of improving antibody reproducibility issues.The study was an evaluation of twenty commercial antibodies for the protein Huntingtin for use in western blot, immunoprecipitation, and immunofluorescence.
The study was well designed and the results were to a high technical standard and quality.The data is presented clearly and looks robust.
It was reassuring to see that a committee of industry and academic representatives had endorsed the platform used.The platform consisted of identification of human cell lines suitable for antibody characterisation studies i.e. with adequate levels of Huntingtin, followed by the development of and contribution of isogenic knockout control cell lines, which were validated at the protein level.The final step was a series of antibody characterisation procedures, limited to the most common research uses of antibodies.Limitations of the study are clearly stated.Two minor errors were spotted.There is an inconsistency between the text and figure 3, where they mention IP was done on twenty antibodies in the text, but results for only ten antibodies are presented in the figure.Perhaps some additional text on why only ten were presented would be of use, or amend the text to reflect what is presented in the figure .A second error is that in the final results paragraph on immunofluorescence, figure 3 is cited, and linked back to, instead of figure 4.
Overall, this will be a highly useful resource for scientists in the HD research community.

Jieya Shao
Washington University in St Louis, St. Louis, Missouri, USA In this study, the authors performed comprehensive analysis of twenty commercially available HTT antibodies for their suitability and performance in Western blot, immunoprecipitation, and immunofluorescence staining applications.The experimental approaches were logically and thoroughly designed.Data are of high-quality and clearly presented.The results will be very helpful for researchers in the HTT field.However, there seems to be two small errors.First, in Figure 3, only the IP data of 10 antibodies were shown while in the text it was stated that 20 antibodies were analyzed.Second, on Page 5, in the last sentence of the second paragraph, the authors seem to cite Figure 3 by mistake.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Cell biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com

Figure 2 .
Figure 2. Huntingtin western blot on various cell lysates.Lysates of WT and HTT KO in DMS 53, HEK 293T and HAP1 were prepared, and 30 μg of protein was processed for western blot with the indicated Huntingtin antibodies ab45169** at 1/5000 and GTX638832** at 1/500.Tris-Glycine 4-20% gels were used for SDS-PAGE.The Ponceau stained transfer is shown as a loading control.Predicted band size: 347 kDa.**Recombinant antibody.

Table 1 .
Summary of the cell lines used.

Is the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
https://doi.org/10.5256/f1000research.168592.r315014© 2024 Shao J.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.